Antimicrobial-antibiofilm compositions and methods of use thereof

ABSTRACT

Compositions comprising chelating agents, metal ion salts, gelling agents or a buffer, antimicrobials, antibiofilm agents and a pH adjuster or a buffer for the prevention and treatment of wound infections and food-borne diseases involving bacterial biofilms are disclosed. The anti-infective properties of a composition include reduction or killing of anaerobic/aerobic/facultative gram-negative and gram-positive wound infection associated bacteria occurring in polymicrobial biofilms. The composition may be in the form of lotion, cream, ointment, dressing, bandage, rinse, soak, gel, spray, or other suitable forms, including certain devices. Additionally, the invention offers an efficient method of delivering the formulated composition containing one or two chelating agents or chelating agents alone or in combination with a metal ion salt using either a nanoparticle or other efficient delivery systems.

CROSS-REFERENCE TO RELATED APPLICATION

This patent/application is a continuation-in-part of U.S. patentapplication Ser. No. 14/772,956, filed Sep. 4, 2015, and entitled“ANTIMICROBIAL-ANTIBIOFILM COMPOSITIONS AND METHODS OF USE THEREOF,”which claims the benefit of and priority to U.S. Provisional PatentApplication No. 61/773,912, filed on Mar. 7, 2013 entitled“ANTIMICROBIAL COMPOSITIONS AND METHODS AND USES THEREOF FOR TREATINGAND PREVENTING WOUND INFECTIONS”; International Patent Application No.PCT/CA2013/050324 filed Apr. 26, 2013 entitled“ANTIMICROBIAL-ANTIBIOFILM COMPOSITIONS AND METHODS OF USE THEREOF”; andU.S. Provisional Patent Application No. 61/834,654 filed Jun. 13, 2013entitled “ANTIMICROBIAL-ANTIBIOFILM COMPOSITIONS AND METHODS OF USETHEREOF”. The content of the above-identified patent applications ishereby expressly incorporated by reference into the detailed descriptionhereof.

FIELD OF THE INVENTION

This invention may relate to methods of using antimicrobial andantibiofilm compositions for prevention and treatment of woundinfections. It further may relate to methods of formulating thecompositions comprising chelating agents, zinc salts, antimicrobials andpharmaceutically acceptable excipients for applications in wound care,disinfectants, cosmetics and medical instruments/devices. Moreparticularly, the invention may relate to an efficient method ofdelivering a pharmaceutically acceptable formulation containing two ormore chelating agents and a zinc salt.

BACKGROUND OF THE INVENTION

From a microbiological perspective, the primary function of normal,intact human and animal skin is to control microbial populations thatlive on the skin surface and to prevent underlying tissue from becomingcolonized and invaded by potential pathogens. Exposure of subcutaneoustissue (i.e. a wound) provides a moist, warm and nutritious environmentthat is conducive to microbial colonization and proliferation.

Since wound colonization is mostly polymicrobial, involving numerousmicroorganisms that are potentially pathogenic, any wound is at somerisk of becoming infected. In the event of an infection a wound fails toheal, the patient suffers increased trauma as well as increasedtreatment costs. General wound management practices become more resourcedemanding. Over 2% of the US population suffers from such chronic,non-healing wounds and it costs the US health care system $20 billion ayear. Wounds are an enormous problem worldwide in humans as well as inanimals.

Thus, concern among health care practitioners regarding the risk ofwound infection is justifiable not only in terms of increased trauma tothe patient but also in view of its burden on financial resources andthe increasing requirement for cost-effective management within thehealth care system. Most wound infections are caused by Staphylococcusaureus (20%), Staphylococcus epidermidis (14%), Enterococci spp. (12%),Escherichia coli (8%), Pseudomonas aeruginosa (8%), Enterobacter spp.(7%), Proteus spp. (3%), Klebsiella pneumoniae (3%), Streptococci spp.(3%) and Candida albicans (3%).

Wounds often have multiple barriers to healing. Wound healing andinfection is influenced by the relationship between the ability ofbacteria to create a stable, prosperous community within a woundenvironment and the ability of the host to control the bacterialcommunity. Since bacteria are rapidly able to form their own protectivemicroenvironment (biofilm) following their attachment to a surface, theability of the host to control these organisms is likely to decrease asthe biofilm community matures. Within a stable biofilm community,interactions between aerobic and anaerobic bacteria are likely toincrease their net pathogenic effect, enhancing their potential to causeinfection and delay healing. Over the last few years, some have linkedbiofilm to chronic wounds. Microscopic evaluation or chronic woundsshowed well organized biofilm with extracellular polymeric substanceadhered around colony bacteria in at least 60% of the chronic wounds.

In recent years, there have been numerous efforts to use antibiotics andantimicrobials for the treatment of non-healing, clinically infectedwounds in humans as well as in animals. These antimicrobial agents areof varying chemical compositions and can include peptides, antiseptics(U.S. Pat. No. 6,700,032), antibiotics, silver ions/compounds (US patentappl. pub. no. 2005/0035327), chitosan (US patent appl. pub. no.2006/0210613; U.S. Pat. No. 6,998,509), nitrofurazone, bismuth thiols,and xylitol (WO 2005/058381).

There have been various attempts by others to create wound care devicessuch as dressings or bandages, gels and ointments comprisingantimicrobial agents. For example, U.S. Pat. No. 3,930,000 discloses theuse of a silver-zinc-allantoinate cream for killing. From amicrobiological perspective, the primary function of normal, intacthuman and animal skin is to control microbial populations that live onthe skin surface and to prevent underlying tissue from becomingcolonized and invaded by potential pathogens. Exposure of subcutaneoustissue (i.e. a wound) provides a moist, warm and nutritious environmentthat is conducive to microbial colonization and proliferation.

Historically, it has been presumed that the properties of bacteria thatcause chronic infections were similar to those of bacteria grownsuspended in liquid growth media. However, research over the past 20years has indicated that many chronic infections are the result of thebiofilm mode of microbial growth. Bacteria in biofilms can be 100 to1000 times more resistant to antibiotics/antimicrobials compared totheir planktonic counterparts. Recent studies have demonstrated biofilmas a potential reason why chronic wounds do not heal (Singh and Barbul,Wound Rep Reg. 16: 1, 2008). In addition, James et al. (Wound Rep Reg.16: 37-44, 2008) has recently demonstrated biofilms in over 60° % ofbacterial infections associated with chronic wounds such as diabeticfoot ulcers, venous leg ulcers and pressure ulcers.

The chronic wound infections are typically persistent infections thatdevelop slowly, seem to be rarely resolved by immune defenses, andrespond transiently to antimicrobial therapy. Thus, there is an unmetclinical need for developing wound care products with both theantibiofilm and antimicrobial activity for prevention and treatment ofacute as well as chronic wounds that involve biofilms. A compositionwith both the antibiofilm and antimicrobial activity kills biofilmbacteria that are highly resistant to antibiotics/antimicrobials and tobody's immune system by inhibiting biofilm formation and/or bydisrupting preformed biofilms. Furthermore, there is also a need for anon-antibiotic wound care or disinfectant composition comprisinggenerally recognized as safe (GRAS).

In an abattoir or meat processing plant, there is a problem ofcontamination of meat and apparatuses (e.g. mincing machine, cutters,slicers, mixers, fillers, or the like) with food poisoning microbials.In a conventional meat processing plant, sodium hypochlorite is used asan anti-microbial during a sterilizing process of meat. Meat such ascarcasses is immersed in a solution of sodium hypochlorite for a certaintime. However, there is a problem of safety for a human body of reactionproducts of sodium hypochlorite adhering to meat.

Thus, there is a need for an anti-microbial for sterilization of meat,with a high degree of safety for humans, and long lasting anti-microbialpower. Such an anti-microbial will keep meat fresher longer and decreaseor prevent degradation of products.

SUMMARY OF THE INVENTION

The instant invention may provide compositions and methods forprevention, decontamination or treatment of acute and chronic woundinfections, or disinfection of fruits, vegetables, meat products, meatand food processing facilities

One embodiment of the invention may provide a composition comprising (a)one or more chelating agents, and (b) one or two metal ion salts.

In another embodiment, a composition of the invention comprises: (a) asmall amount of at least two chelating agents, (b) a small amount of atleast one metal ion salt, wherein the amount of each of components (a)and (b) is sufficient to form an effective anti-infective compositionagainst bacterial infections in wounds and for application asdisinfectants.

In yet another embodiment, a composition of the invention comprises: (a)a small amount of at least two chelating agents, (b) a small amount ofat least one metal ion salt, and (c) pharmaceutically acceptableexcipients.

Still another embodiment of the invention may provide an anti-infectivecomposition comprising two chelating agents and one or two metal ionsalts that are effective against bacteria and fungi causing woundinfections (infections of cuts, bruises, surgical sites, lacerations,abrasions, punctures, incisions, gunshots, burns, pyoderma, atopicdermatitis, eczema, pressure ulcers, venous and artery leg ulcersdiabetic foot ulcers, etc.), cystic fibrosis (CF)-associated infections,community or hospital acquired infections or food-borne diseases.

The compositions of the invention may be for use against one or moreinfection-associated bacteria or yeasts selected from the groupconsisting of Methicillin-resistant Staphylococcus aureus (MRSA).Staphylococcus epidermidis, Coagulase negative staphylococci (CoNS),Vancomycin resistant Enterococci (VRE), Carbapenem resistant Klebsiellapneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Methicllinresistant Staphylococcus pseudintermedius (MRSP), Malasseziapachydermatis, Salmonella typhimurium, Escherichia coli O157:H7, Candidaalbicans, Listeria monocytogenes, Campylobacter jejuni, Bacillus spp.,Streptococcus agalactiae, Streptococcus uberis, Esherichia coli,Salmonella choleraesuis, Stenotrophomonas maltophlla, Enterococcusfaecalis, Proteus mirabilis, Klebsiella spp., Enterobacter spp., andCitrobacter spp.

A further embodiment of the invention may provide an anti-infectivecomposition comprising at least two chelating agents and one or twometal ion salts that are for use against veterinary wound infections, ormastitis and otitis associated bacteria and yeasts.

In an embodiment, the chelating agent is between about 5000 mg/L andabout 50000/L of the composition. In an embodiment, the metal ion saltis between about 1000 mg/L and about 10000 mg/L of the composition.

The chelating agents may be selected from the group consisting of EDTA,EGTA, DTPA, EDDHA, IDA, CDTA, HEDTA, HEIDA, NTA, sodium citrate,potassium citrate, ovotransferrin and lactoferrin. The metal ion saltsmay be selected from the group consisting of zinc chloride, zinclactate, zinc citrate, zinc gluconate, zinc sulfate zinc acetate, silverion or silver sulfadiazine, silver sulfate, silver nitrate, and silvercarbonate.

In another embodiment, the chelating agents are EDTA and sodium citrate,and metal ion salt is zinc chloride or zinc sulfate. The EDTA may bepresent at about 10 mg/ml and sodium citrate may be present at about 10mg/ml. The zinc chloride or zinc lactate may be present at about 1mg/ml.

The composition may further comprise one or more ingredients selectedfrom the group consisting of: water, citrate buffer, citric acid,stabilizing agent, a flavoring agent, vitamins, minerals, herbals, asurfactant, an antimicrobial peptide, an antimicrobial and a pHadjuster.

The invention may also teach methods of preparing a suitable formulationfor wound care application in a variety of ways, for example in adisinfecting solution, a lotion, cream, a gel, a spray, athermo-reversible gel spray, a paste, a balm, a bandage, a dressing, agauze, a wound irrigating device, a wrap, and mastitis teat dipsolutions.

The invention further may teach methods of preparing suitableformulations for disinfectants and cosmetics. The disinfectants haveapplications in disinfecting fruits, vegetables, food and meatprocessing facilities, hospitals, barns, medical instruments, and otherindustrial and institutional facilities. The cosmetics include shampoosand antimicrobial body lotions and creams.

The invention further may teach methods of preventing or treating meatspoilage comprising topical use of the composition on meat or meat ormeat products. The method may include one or more of coating, spraying,misting, injecting, soaking, flushing, dipping and rinsing.

The formulations can also include natural or synthetic flavorings andcoloring agents. Thickening agents can also be added to compositions ofthe invention such as guar gum, carbopol, polyethylene glycol, pluronicF-127, sodium alginate, carboxymethyl cellulose, xanthan gum and otherpharmaceutically acceptable thickening agents.

Other formulations will be readily apparent to one skilled in the art. Acomposition of the invention can include antibiofilm enzymes (cellulase,beta-N-acetylgluconase, DispersinB, papain, DNase 1, etc.),antimicrobial peptides, antibiotics (gentamicin, ciprofloxacin,ampicillin, cefamandole nafate, rifampicin, etc.), antimicrobials(triclosan, chlorhexidine, quaternary ammonium compounds, silver, silversalts, etc.) and other antibiofilm compounds.

The invention may also teach the use of liposomal or nanoparticledelivery systems that enhance the stability and efficacy ofanti-infective compounds in the compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone and in combinationon methicillin-resistant Staphylococcus aureus (MRSA) growth and biofilmformation.

FIG. 2 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.1 mg/ml) alone and incombination on methicillin-resistant Staphylococcus pseudintermedius(MRSP) growth and biofilm formation.

FIG. 3 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone and in combinationon Pseudomonas aeruginosa growth and biofilm formation.

FIG. 4 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.062 mg/ml) and ZnCl₂ (0.1 mg/ml) alone and incombination on Listeria monocytogenes growth and biofilm formation.

FIG. 5 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂ and EDTA+ZnCl₂ combinations onmethicillin-resistant Staphylococcus aureus (MRSA) growth and biofilmformation.

FIG. 6 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onmethicillin-resistant Staphylococcus pseudintermedius (MRSP) growth andbiofilm formation.

FIG. 7 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.25 mg/ml), and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onPseudomonas aeruginosa growth and biofilm formation.

FIG. 8 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (025 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onSalmonella choleraesuis ATCC 10708 growth and biofilm formation.

FIG. 9 is a bar graph showing the inhibitory effect of sodium citrate (3mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onEscherichia coli O157:H7 growth and biofilm formation.

FIG. 10 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and incombination on Escherichia coli O157:H7 growth and biofilm formation.

FIG. 11 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onStaphylococcus epidermidis growth and biofilm formation.

FIG. 12 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onCoagulase-negative Staphylococci (CoNS-42) growth and biofilm formation.

FIG. 13 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onStreptococcus agalactiae ATCC 12386 growth and biofilm formation.

FIG. 14 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onKlebsiella pneumoniae growth and biofilm formation.

FIG. 15 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onAcinetobacter baumannii growth and biofilm formation.

FIG. 16 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onStenotrophomonas maltophilia growth and biofilm formation.

FIG. 17 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onVancomycin-resistant Enterococci (VRE) growth and biofilm formation.

FIG. 18 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onEnterococcus faecalis growth and biofilm formation.

FIG. 19 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onProteus mirabilis growth and biofilm formation.

FIG. 20 is a bar graph showing the inhibitory effect of sodium citrate(3 mg/ml), EDTA (0.25 mg/ml) and ZnCl₂ (0.1 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onCandida albicans growth and biofilm formation.

FIG. 21 is a bar graph showing the inhibitory effect of sodium citrate(1.5 mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.05 mg/ml) alone, and sodiumcitrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations onMalassezia pachydermatis growth and biofilm formation.

FIG. 22 is a bar graph showing the inhibitory effect of sodium citrate(1.5 mg/ml), EDTA (0.125 mg/ml) and ZnCl₂ (0.05 mg/ml) alone and incombination on Malassezia pachydermatis growth and biofilm formation.

FIG. 23 is a bar graph showing the inhibitory effect of sodium citrate(1.6 mg/ml), and EDTA (0.5 mg/ml) alone and in combination on Malasseziapachydermatis biofilm formation.

FIG. 24 is a bar graph showing the inhibitory effect of sodium citrate(3.2 and 6.4 mg/ml), and EDTA (0.5 mg/ml) alone and in combination onStaphylococcus aureus biofilm formation.

FIG. 25 is a bar graph showing the inhibitory effect of sodium citrate(3.2 and 6.4 mg/ml), and EDTA (0.5 mg/ml) alone and in combination onMalassezia furfur biofilm formation.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antimicrobial” refers to a compound or a composition thatkills or inhibits or stops the growth of microorganisms, including, butnot limited to bacteria and yeasts.

The term “biofilm” refers to a structured community of microorganismsenclosed in a self-produced extracellular polymeric matrix, and attachedto a biotic or abiotic surface. Bacteria in a biofilm can be 1000 timesmore resistant to antibiotics/antimicrobials compared to theirplanktonic (free living) counterparts.

The term “biofilm formation” refers to the attachment of microorganismsto surfaces and the subsequent development of multiple layers of cells.

The term “antibiofilm” refers to inhibition of microbial biofilmformation and disruption or dispersal of preformed biofilms.

The term “infection” refers to the invasion and multiplication ofmicroorganisms such as bacteria, viruses, and parasites that are notnormally present within the body. An infection may cause no symptoms andbe subclinical, or it may cause symptoms and be clinically apparent. Aninfection may remain localized, or it may spread through the blood orlymphatic vessels to become systemic (body wide). Microorganisms thatlive naturally in the body are not considered infections.

The term “wound” refers to a type of injury in which skin is torn, cut,or punctured (an open wound), or where blunt force trauma causes acontusion (a closed wound). In pathology, it specifically refers to asharp injury, damages to the dermis of the skin.

The term “acute wound” refers to those that are new and in the firstphase of healing. Acute wounds are characterized by skin layers thathave been punctured or broken through by an external force or object.Any acute wound can progress to a chronic wound if it does not healwithin the expected time frame or as a result of poor blood supply,oxygen, nutrients or hygiene. Acute wounds should be properly treated toavoid infection, inflammation or constant pressure. Acute wounds arecategorized based on causes such as lacerations, abrasions, punctures,incisions, gunshots, burns, and type according to the size and depth(superficial or deep).

The term “chronic wound” refers to a wound that just will not repairitself over time. Chronic wounds are often thought to be “stuck” in oneof the phases of wound healing, and are most often seen in the olderadult population. Typically, if a wound is not healing as expectedwithin 2-3 months, it is considered chronic. Chronic wounds includepressure ulcers (e.g. bed sores), arterial and venous leg ulcers, anddiabetic ulcers.

The term “disinfectants” refers to substances that are applied tonon-living objects to destroy microorganisms that are living on theobjects. Disinfection does not necessarily kill all microorganisms,especially resistant bacterial spores; it is less effective thansterilization, which is an extreme physical and/or chemical process thatkills all types of life. Disinfectants are different from otherantimicrobial agents such as antibiotics, which destroy microorganismswithin the body, and antiseptics, which destroy microorganisms on livingtissue. Disinfectants are also different from biocides—the latter areintended to destroy all forms of life, not just microorganisms.Disinfectants work by destroying the cell wall/membrane of microbes orinterfering with metabolism and growth.

The term “inhibition” refers to at least a decrease of wound-associatedbacterial growth and biofilm formation.

The term “mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports or pet animals, such as dogs, horses, cats, cattle, pigs,sheep, etc.

The term “prevention” refers to at least preventing a conditionassociated with bacteria occurring in a mammal, particularly when themammal is found to be predisposed to having the condition but has notyet been diagnosed as having it.

The term “subject” refers to a living vertebrate such as mammal(preferably human and pet animals) in need of treatment.

The term “therapeutically effective amount” refers to a quantity of acomposition high enough to provide a significant positive modificationof the subject's condition(s) to be treated.

A “preventative amount” as used herein includes a prophylactic amount,for example, an amount effective for preventing or protecting againstwounds, skin infections and related diseases, and symptoms thereof, andamounts effective for alleviating or healing wounds, skin infections,related diseases, and symptoms thereof. By administering a peptidesuitable for use in methods of the invention concurrently with anantimicrobial, the peptide and/or the antimicrobial may be administeredin a dosage amount that is less than the dosage amount required when theantimicrobial is administered as a sole active ingredient. Byadministering lower dosage amounts of active ingredient, side effectsassociated therewith could be reduced.

The term “treatment” refers to an intervention performed with theintention of preventing the further development or altering thepathology of an existing disorder. Accordingly, “treatment” refers toboth therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already with the infection aswell as those in which the infection is to be prevented. In regards, towound infections, “treating or treatment” is intended to mean at leastthe mitigation of wound healing conditions associated with bacterialinfections in a subject, such as a mammal, including but not limited to,a human, that is affected at least in part by the condition, andincludes, but is not limited to, modulating, inhibiting the condition,and/or alleviating the condition.

The term “metal ion salt” refers to salt of a metal ion such as zincchloride, zinc lactate, zinc citrate, zinc gluconate, zinc sulfate zincacetate, silver ion or silver sulfadiazine, silver sulfate, silvernitrate, and silver carbonate.

The present invention may teach anti-infective compositions offeringantimicrobials and antibiofilm activity, containing combinations ofchelating agents with other antimicrobial agents, such as, for example,antimicrobials/antibiofilm compounds, metal ion salts with gellingagents, surfactants or stabilizing agents.

Novel compositions that combine chelating agents together with metal ionsalts such that lesser quantities of chelating agents and/or metal ionsalts than would normally be necessary for an antimicrobial compositionare used to achieve significant bacterial growth and biofilm inhibition.Higher concentrations of these compounds can be used if it is desiredfor certain applications.

The amount of EDTA to be used in the antimicrobial composition of thisinvention can be between 10000 to 100000 mg/L. The higher end of thisstated range might be used to prepare a concentrated product that wouldbe diluted prior to use. For non-concentrated products, the amount of tobe used in this invention is preferably between about 5000 to 10000mg/L. Preferably, the range is between about 1000 to 5000 mg/L, morepreferably 0.1 to 1 mg/ml.

The amount of citrate to be used should be between about 1000 to 5000mg/L. The higher end of this range might apply if the compositions wereformulated as a concentrate. For non-concentrated products, the amountof chelating agent to be used in this invention is preferably betweenabout 500 to 5000 mg/L. Preferably, the range is between about 1000 to3000 mg/L, more preferably between about 2000 to 3000 mg/L, and morepreferably 1 to 10 mg/ml.

Preparation

By one method, if a two-component composition is formed containing oneor two chelating agents and a metal ion salt, these compounds can becombined in the following manner. With good stirring, a chelating agentcan be dissolved in water, followed by a metal ion salt. It should benoted, however, that the addition order can be reversed.

Additionally, antimicrobials/antimicrobial peptides, antibiotics,antibiofilm compounds, quaternary ammonium compounds and surfactantsalso may be advantageously combined with chelating agents in anantimicrobial composition. A composition of the invention comprises: (a)a small amount of at least one or two chelating agent; (b) a smallamount of a metal ion salt or iron-sequestering glycoprotein orantimicrobial peptide or an antibiotic or an antibiofilm compound; and(c) a sparing amount of at least one compound from the group consistingof a stabilizing agent and/or a gelling agent and/or a surfactant,wherein, the amount of each of component (a), (b) and (c) is sufficientto form, in combination, an effective anti-infective composition forprevention and treatment of acute and chronic wound infections(infections of cuts, bruises, surgical sites, lacerations, abrasions,punctures, incisions, gunshots, burns, pyoderma, atopic dermatitis,eczema, pressure ulcers, venous and artery leg ulcers diabetic footulcers, etc).

The concentration of active components in the compositions may vary asdesired or necessary to decrease the amount of time the composition ofthe invention is used to prevent or treat wound infections and fordisinfection. These variations in active components concentration areeasily determined by persons skilled in the art.

Compositions

The present invention may include unique and enhanced anti-infectivecompositions for the prevention and treatment of wound infectionscomprising at least two chelating agents and one metal ion salt.

In an embodiment, two chelating agents and a metal ion salt containingcomposition includes an antimicrobial compound. The chelating agents anda metal ion salt containing composition with an antimicrobialand/antibiofilm compound has an enhanced inhibitory effect on woundinfection-associated bacterial growth and biofilm formation.Furthermore, addition of an antimicrobial compound to a compositioncontaining chelating agents and a metal ion salt can make thecomposition effective against pathogens associated with wound infectionsand microbial contamination causing food-borne diseases.

In an embodiment of the invention, an enhanced antimicrobial-antibiofilmcomposition comprises at least one or two chelating agents, one metalion salt and one or more antimicrobial agents comprising antiseptics(e.g., triclosan, chlorhexidine salt, cetylpyridinium chloride, etc.),antibiotics and bacteriocins (e.g., nisin, epidermin, gallidennin,cinnamycin, duramycin, lacticin 481, etc.), and iron-sequesteringglycoproteins (ovotransferrin, lactoferrin and serrotransferrin).Additionally, the wound care or disinfectant compositions may compriseingredients such as citrate (e.g., citric acid, zinc citrate, sodiumcitrate, potassium citrate, etc.), minerals (e.g., mineral salts such aszinc chloride, zinc gluconate, zinc lactate, zinc citrate, zinc sulfate,zinc acetate, silver, silver sulfate, silver sulfadiazine, silvernitrate, silver carbonate, etc.), and triterpenoids (e.g., oleanolicacid and ursolic acid) and chitosan

In an embodiment, a composition comprises an antibiotic and one or twochelating agents and also one metal ion salt. Antibiotics are wellknown. Groups of antibiotics include, but are not limited to, β-lactaminhibitors (e.g., penicillin, ampicillin, amoxicillin, methicillin,etc.), cephalosporins (e.g., cephalothin, cephamycin, etc.),aminoglycosides (e.g., streptomycin, tobramycin, etc.), polyenes (e.g.,amphotericin, nystatin, etc.), macrolides (e.g., erythromycin, etc.),tetracyclines (e.g., tetracycline, doxycycline, etc.), nitroimidazole(e.g., metronidazole), quinolones (e.g., nalidixic acid), rifamycins(e.g., rifampin), and sulfonamides (e.g., sulfanilamide), nitroaromatics(e.g., chloramphenicol) and pyridines (e.g., isoniazid).

In an embodiment, a composition comprises an antiseptic, one or twochelating agents and one metal ion salt. Antiseptics are agents thatkill or inhibit the growth of microorganisms on the external surfaces ofthe body. Antiseptics include, but are not limited to, triclosan,chlorhexidine salt, and cetylpyridinium chloride.

In an embodiment, a composition comprises an antibiofilm compound, oneor two chelating agents and a metal ion salt. Antibiofilm compoundsinclude, but not limited to, DispersinB, DNase I, Proteinase K, apyrase,cis-2-decnoic acid, alginate lyase, lactoferrin, gallium, cellulase, and5-fluorouracil.

In an embodiment, a composition is effective for inhibiting growth andbiofilm formation in wound infection and food-borne disease associatedbacteria. The composition is also effective in disrupting or dispersingpreformed biofilms, which makes biofilm-embedded bacteria moresusceptible to antimicrobial killing. Under appropriate environmentalconditions, such as moisture and pH, infections can be modulated usingembodiments of the invention.

An embodiment of the invention may also include other pharmaceuticallyacceptable vehicles, diluents, and additives such as antioxidants,anti-inflammatory compounds, vitamins, tissue degrading enzymes, buffersand solutes that render the formulation isotonic in the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents, surfactants and thickening agents.

Wound Care Formulations

A composition of the invention may be added to a variety of formulationssuitable for applying/delivering the composition to wounds, including,but not limited to, disinfecting solutions, lotions, creams, gels,sprays, gel spray, bandage, dressings, wraps, gauze, tapes, adhesivesand wound irrigation devices. To provide such formulations, acomposition of this invention is combined with one or morepharmaceutically acceptable excipients.

Formulations including, but not limited to, pharmaceutically acceptablecompositions comprising one or two chelating agents and a metal ion saltin combination with an antiseptic, an antibiotic, an antimicrobial, aniron-sequestering glycoprotein, a bacteriocin, extracellular matrix orchitosan can be prepared by any known method.

In general, methods of manufacturing anti-infective compositions maycomprise combining a pharmaceutically acceptable carrier and aneffective amount of both chelating agents and a metal ion salt with anantiseptic, an antibiotic, a bacteriocin, an antimicrobial peptide orchitosan.

A variety of carriers and excipients can be used to formulate anembodiment of this invention and are well known. Such pharmaceuticallyacceptable vehicles include, but are not limited to, water, ethanol,humectants such as polypropylene glycol, glycerol and sorbitol, gellingagents such as cellulose derivatives, polyoxypropylene/polyoxyethyleneblock copolymers, carboxy methyl cellulose, pluronic F-127, sodiumalginate, polyethylene glycol, thickening agents such as Carbopol™ 934.

Method of Treatment

Another aspect of this invention may include a method for treating woundinfections, and also for decontaminating wound surfaces as well asfood/meat processing facilities. In general, wound infections may betreated by applying to the infected wound of a subject with an effectiveamount of one or more chelating agents and a metal ion salt incombination with one or more antimicrobial agent effective to reducewound infections.

Before selling meat such as chicken, beef and pork for consumption, itis necessary to stop or retard the growth of pathogenic microorganismsand it is preferable to kill pathogenic microorganisms such as bacteriawhich may cause food poisoning due to their presence in the meat. Thus,the invention provides a method of preventing or treating meat spoilagecomprising topical use of the composition of the invention on meat ormeat products. Meat or meat products may be one or more of beef, pork,lamb, goat, horse, chicken and fish. The meat or meat products mayinclude intestine or intestinal parts of pigs, cattle, sheep, goats andhorses used for making sausage casings. They may further includecollagen or cellulose used for making artificial sausage casings

The compositions may be applied by one or more of coating, spraying,misting, injecting, soaking, flushing, dipping and rinsing. The flushingmay include flushing water lines or meat processing lines and cleaningequipment in meat processing and packaging plants.

For use in treating or disinfecting meat, preferred concentration rangeof ingredients may include:

-   -   (i) Sodium Citrate: (a) 50,000 mg/L-100,000 mg/L, (b) 25,000        mg/L-50,000 mg/L, (c) 10,000 mg/L-25,000 mg/L, (d) 5,000        mg/L-10,000 mg/L, & (e) 1,000 mg/L-5,000 mg/L.    -   (ii) Disodium EDTA: (a) 10,000 mg/L-25,000 mg/L, (b) 5,000        mg/L-10,000 mg/L, (c) 1,000 mg/L-5,000 mg/L, (d) 100 mg/L-1,000        mg/L, & (e) 100 mg/L-500 mg/L    -   (iii) Zinc Chloride: (a) 1,000 mg/L-5,000 mg/L, (b) 500        mg/L-1,000 mg/L, (c) 100 mg/L-500 mg/L, and (d) 10 mg/L-100        mg/L.

In one embodiment, one or more chelating agents and a metal ion salttogether is formulated as pharmaceutically acceptable medicament asdescribed herein comprising a carrier and an effective amount ofcomposition comprising one or more chelating agents and a metal ion saltas active ingredients.

An exemplary dosing regime of a wound care composition of this inventionis application of a composition to the wound surface of a subject(animal or human) at least once or twice. According to this embodiment,a subject would apply a composition of the invention to the woundsurface from one to three times daily depending on the type of wound andseverity of infection. For animals or pets, the composition of theinvention can be used as a lotion or a cream, or a gel or a spray or adressing twice or thrice a day.

In a further embodiment of the invention, an enhanced woundanti-infective composition does not present any antibiotic resistanceconcerns and bio-compatibility/safety issues. Also, the composition ofthis invention comprising one or two chelating agents (EDTA and sodiumcitrate) and a metal ion salt (zinc chloride or zinc sulfate or zinclactate) has GRAS (Generally Recognized as Safe) status and all theseingredients are food as well as feed additives.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example 1: Inhibitory Effect of Sodium Citrate, EDTA and ZincChloride Alone and in Combination on Methicillin-ResistantStaphylococcus aureus (MRSA) Growth and Biofilm Formation

An overnight broth culture of S. aureus was grown in TSB and used asinoculum. 96-well microplates containing TSB in the absence and thepresence of each compound (sodium citrate or EDTA or Zinc chloride)separately and together (Sodium chloride+EDTA+Zinc chloride) wereinoculated and incubated at 37° C. for 24 hours. Growth of planktoniccells based on absorbance at 600 nm using Labsystems Multiskan Ascentmicroplate reader was determined. Biofilm was measured by discarding themedia in the wells, rinsing the well three times with water, andstaining the bound cells with crystal violet. The dye was thensolubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition, comprisingsodium citrate, EDTA and zinc chloride showed an enhanced inhibitoryeffect on biofilm formation, as compared to sodium citrate, or EDTA, orzinc chloride alone (FIG. 1).

Example 2: Inhibitory Effect of Sodium Citrate, EDTA and Zinc ChlorideAlone, and in Combination on Methicillin-Resistant Staphylococcuspseudintermedius (MRSP) Growth and Biofilm Formation

An overnight broth culture of methicillin resistant S. pseudintermediuswas grown in TSB and used as inoculum. 96-well microplates containingTSB in the absence and the presence of each compound (sodium citrate orEDTA or Zinc chloride) separately and together (Sodiumchloride+EDTA+Zinc chloride) were inoculated and incubated at 37° C. for24 hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition, comprising sodium citrate, EDTA and zinc chloride showed anenhanced inhibitory effect on biofilm formation, as compared to sodiumcitrate, or EDTA, or zinc chloride alone (FIG. 2).

Example 3: Inhibitory Effect of Sodium Citrate, EDTA, and Zinc ChlorideAlone, and in Combination on Pseudomonas aeruginosa Growth and BiofilmFormation

An overnight broth culture of P. aeruginosa was grown in TSB and used asinoculum. 96-well microplates containing TSB in the absence and thepresence of each compound (sodium citrate or EDTA or Zinc chloride)separately and together (Sodium chloride+EDTA+Zinc chloride) wereinoculated and incubated at 37° C. for 24 hours. Growth of planktoniccells based on absorbance at 600 nm using Labsystems Multiskan Ascentmicroplate reader was determined. Biofilm was measured by discarding themedia in the wells, rinsing the well three times with water, andstaining the bound cells with crystal violet. The dye was thensolubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition, comprisingsodium citrate, EDTA and zinc chloride showed an enhanced inhibitoryeffect on biofilm formation, as compared to sodium citrate, or EDTA, orzinc chloride alone (FIG. 3).

Example 4: Inhibitory Effect of Sodium Citrate, EDTA, and Zinc ChlorideAlone and in Combination on Listeria monocytogenes Growth and BiofilmFormation

An overnight broth culture of L. monocytogenes was grown in TSB and usedas inoculum. 96-well microplates containing TSB in the absence and thepresence of each compound (sodium citrate or EDTA or Zinc chloride)separately and together (Sodium chloride+EDTA+Zinc chloride) wereinoculated and incubated at 37° C. for 24 hours. Growth of planktoniccells based on absorbance at 600 nm using Labsystems Multiskan Ascentmicroplate reader was determined. Biofilm was measured by discarding themedia in the wells, rinsing the well three times with water, andstaining the bound cells with crystal violet. The dye was thensolubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition, comprisingsodium citrate, EDTA and zinc chloride showed an enhanced inhibitoryeffect on biofilm formation, as compared to sodium citrate, or EDTA, orzinc chloride alone (FIG. 4).

Example 5: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Methicillin-Resistant Staphylococcus aureus [MRSA]Growth and Biofilm Formation

An overnight broth culture of S. aureus (MRSA) was grown in TSB and usedas inoculum. 96-well microplates containing TSB in the absence and thepresence of each compound (sodium citrate or EDTA or Zinc chloride)separately and Sodium citrate+EDTA, Sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA and Sodium citrate+ZnCl₂ combinations showed anenhanced inhibitory effect on biofilm formation, as compared to sodiumcitrate, or EDTA, or zinc chloride alone (FIG. 5).

Example 6: Effect of Sodium Citrate, EDTA, and ZnCl₂ Alone, and SodiumCitrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂ Combinations onMethicillin Resistant Staphylococcus pseudintermedius (MRSP) Growth andBiofilm Formation

An overnight broth culture of MRSP was grown in TSB and used asinoculum. 96-well microplates containing TSB in the absence and thepresence of each compound (sodium citrate or EDTA or Zinc chloride)separately and Sodium citrate+EDTA, Sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA, Sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinationsshowed an enhanced inhibitory effect on biofilm formation, as comparedto sodium citrate, or EDTA, or zinc chloride alone (FIG. 6).

Example 7: Inhibitory Effect of Sodium Citrate, EDTA, and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂ and EDTA+ZnCl₂Combinations on Pseudomonas aeruginosa Growth and Biofilm Formation

An overnight broth culture of P. aeruginosa was grown in TSB and used asinoculum. 96-well microplates containing TSB in the absence and thepresence of each compound (sodium citrate or EDTA or Zinc chloride)separately and Sodium citrate+EDTA, Sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA, and EDTA+ZnCl₂ combinations showed an enhancedinhibitory effect on biofilm formation, as compared to sodium citrate,or EDTA, or zinc chloride alone (FIG. 7).

Example 8: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Salmonella choleraesuis ATCC 10708

An overnight broth culture of S. choleraesuis ATCC 10708 was grown inTSB and used as inoculum. 96-well microtiter plates containing TSB inthe absence and the presence of each compound (sodium citrate or EDTA orZinc chloride) separately and sodium citrate+EDTA, sodium citrate+ZnCl₂,and EDTA+ZnCl₂ combinations were inoculated and incubated at 37° C. for24 hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.8).

Example 9: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Escherichia coli O157:H7

An overnight broth culture of E. coli O157:H7 was grown in TSB and usedas inoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA, Sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinationsshowed an enhanced inhibitory effect on biofilm formation, as comparedto sodium citrate, or EDTA or zinc chloride alone (FIG. 9).

Example 10: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and in Combination on Escherichia coli O157:H7

An overnight broth culture of E. coli O157:H7 was grown in TSB and usedas inoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and together (Sodium chloride+EDTA+Zinc chloride) wereinoculated and incubated at 37° C. for 24 hours. Growth of planktoniccells based on absorbance at 600 nm using Labsystems Multiskan Ascentmicroplate reader was determined. Biofilm was measured by discarding themedia in the wells, rinsing the well three times with water, andstaining the bound cells with crystal violet. The dye was thensolubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. A composition comprisingsodium citrate, EDTA, and ZnCl₂ showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA, or zincchloride alone (FIG. 10).

Example 11: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Staphylococcus Epidermidis

An overnight broth culture of S. epidermidis was grown in TSB and usedas inoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂ and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.11).

Example 12: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Coagulase-Negative Staphylococci (CoNS-42)

An overnight broth culture of CoNS-42 was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.12).

Example 13: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Streptococcus agalactiae ATCC 12386

An overnight broth culture of S. agalactiae was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.13).

Example 14: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Klebsiella pneumoniae

An overnight broth culture of K. pneumoniae was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.14).

Example 15: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Acinetobacter baumannii

An overnight broth culture of A. baumannii was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.15).

Example 16: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Stenotrophomonas maltophilia

An overnight broth culture of S. maltophilia was grown in TSB and usedas inoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.16).

Example 17: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Vancomycin-Resistant Enterococci (VRE)

An overnight broth culture of VRE was grown in TSB and used as inoculum.96-well microtiter plates containing TSB in the absence and the presenceof each compound (sodium citrate or EDTA or Zinc chloride) separatelyand sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.17).

Example 18: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Enterococcus Faecalis

An overnight broth culture of E. faecalis was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.18).

Example 19: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Proteus Mirabilis

An overnight broth culture of P. mirabilis was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.19).

Example 20: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Candida albicans

An overnight broth culture of C. albicans was grown in TSB and used asinoculum. 96-well microtiter plates containing TSB in the absence andthe presence of each compound (sodium citrate or EDTA or Zinc chloride)separately and sodium citrate+EDTA, sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations were inoculated and incubated at 37° C. for 24 hours.Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. ASodium citrate+EDTA combination showed an enhanced inhibitory effect onbiofilm formation, as compared to sodium citrate, or EDTA alone (FIG.20).

Example 21: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Alone,and Sodium Citrate+EDTA, Sodium Citrate+ZnCl₂, and EDTA+ZnCl₂Combinations on Malassezia pachydermatis

An overnight broth culture of Malassezia pachydermatis was grown inSabouraud Dextrose Broth and used as inoculum. 96-well microtiter platescontaining TSB in the absence and the presence of each compound (sodiumcitrate or EDTA or Zinc chloride) separately and sodium citrate+EDTA,sodium citrate+ZnCl₂, and EDTA+ZnCl₂ combinations were inoculated andincubated at 37° C. for 24 hours. Growth of planktonic cells based onabsorbance at 600 nm using Labsystems Multiskan Ascent microplate readerwas determined. Biofilm was measured by discarding the media in thewells, rinsing the well three times with water, and staining the boundcells with crystal violet. The dye was then solubilized with 33% aceticacid, and absorbance at 630 nm was determined using a microtiter platereader. A Sodium citrate+EDTA, Sodium citrate+ZnCl₂, and EDTA+ZnCl₂combinations showed an enhanced inhibitory effect on biofilm formation,as compared to sodium citrate, or EDTA or zinc chloride alone (FIG. 21).

Example 22: Inhibitory Effect of Sodium Citrate, EDTA and ZnCl₂ Aloneand in Combination on Malassezia pachydermatis

An overnight broth culture of Malassezia pachydermatis was grown inSabouraud Dextrose Broth and used as inoculum. 96-well microtiter platescontaining TSB in the absence and the presence of each compound (sodiumcitrate or EDTA or Zinc chloride) separately and together (Sodiumchloride+EDTA+Zinc chloride) were inoculated and incubated at 37° C. for24 hours. Growth of planktonic cells based on absorbance at 600 nm usingLabsystems Multiskan Ascent microplate reader was determined. Biofilmwas measured by discarding the media in the wells, rinsing the wellthree times with water, and staining the bound cells with crystalviolet. The dye was then solubilized with 33% acetic acid, andabsorbance at 630 nm was determined using a microtiter plate reader. Acomposition comprising sodium citrate, EDTA, and ZnCl₂ showed anenhanced inhibitory effect on biofilm formation, as compared to sodiumcitrate, or EDTA, or zinc chloride alone (FIG. 22).

Example 23: Inhibitory Effect of Sodium Citrate and EDTA Alone and inCombination on Malassezia pachydermatis

An overnight broth culture of Malassezia pachydermatis was grown inSabouraud Dextrose Broth and used as inoculum. 96-well microtiter platescontaining TSB in the absence and the presence of each compound (sodiumcitrate or EDTA) separately and together (Sodium citrate+EDTA) wereinoculated and incubated at 32° C. for 48 hours. Biofilm was measured bydiscarding the media in the wells, rinsing the well three times withwater, and staining the bound cells with crystal violet. The dye wasthen solubilized with 33% acetic acid, and absorbance at 630 nm wasdetermined using a microtiter plate reader. The Experiment was repeatedfour times and the results combined. A composition comprising sodiumcitrate and EDTA showed an enhanced inhibitory effect on biofilmformation at p=0.04, as compared to sodium citrate or EDTA alone (FIG.23). The unit of all concentrations in FIG. 23 is mg/ml.

Example 24: Inhibitory Effect of Sodium Citrate and EDTA Alone and inCombination on Staphylococcus Aureus

A 12-well polystyrene microtiter plate biofilm assay was used todetermine the inhibitory effect of disodium ethylenediaminetetraaceticacid (NaEDTA) and sodium citrate (S. cit) alone and in combination onStaphylococcus aureus. Briefly, Staphylococcus aureus biofilms weredeveloped in a 12-well polystyrene microtiter plate. Initially anovernight culture was grown in TSB for 16 hours at 37° C., the OD₆₀₀ wastaken and the culture was diluted to 10⁴ CFU/ml. To each well either 1ml of water (control) or 1 ml of treatment was added followed by 1 ml ofthe diluted S. aureus cell culture. The 12 well plate was incubated for16 hours at 37° C. The planktonic medium was removed. The wells wererinsed once with 10 mM PBS buffer (pH 7.2) and fresh 10 mM PSB buffer(pH 7.2) added to the wells. The plate was sonicated for 15 seconds. Thewells were scraped and solution removed from the 12 well plate and putinto 10 ml culture tubes. The tubes were vortexed for 30 seconds. 200 μlof solution was transferred from the tubes to a 96 well plate where aserial dilution was done. The dilution series was then plated induplicate onto TSA plates. The plates were incubated for 16 hours at 37°C. Biofilm-embedded viable cells were quantified by colony forming unit(CFU) counts after 16 hours of incubation. The experiment was repeatedtwice and the results are combined. The combination of NaEDTA and sodiumcitrate performed better than NaEDTA or sodium citrate alone. Inparticular, the combination of NaEDTA (0.5 mg/ml) and S. cit (6.4 mg/ml)performed significantly better than NaEDTA at 0.5 mg/ml (p=0.05421333)or sodium citrate at 6.4 mg/ml alone (p=0.01529095), as shown in FIG.24.

Example 25: Inhibitory Effect of Sodium Citrate and EDTA Alone and inCombination on Malassezia furfur

A 12-well polystyrene microtiter plate biofilm assay was used todetermine the inhibitory effect of disodium ethylenediaminetetraacticacid (NaEDTA) and sodium citrate (S. cit) Formulation on Malasseziafurfur. Briefly, Malassezia furfur biofilms were developed on 12-wellpolystyrene microtiter plate to provide a rapid and simple method forassaying biofilm embedded Malassezia furfur. The mDixon agar plate washeavily inoculated with glycerol stock of Malassezia furfur andincubated at 32° C. for 4 days. The colonies were then inoculated in 20mls of mDixon media and incubated at 32° C. for 2 days. The cells werepelleted by centrifuging at 8000 rpm for 10 minutes. The supernatant wasremoved and cells were resuspended in 20 mls of mDixon media. The cellswere diluted to approximately 1×10⁴ cfu/ml. Add to each well of a12-well microtiter plate 1.0 ml water (control) or 1.0 ml of testingagent then add 1.0 ml of the diluted liquid culture to each well of the12-well plate. Incubate the plate at 32° C., rocking at 15 rpm for 24hrs to allow cells to adhere to plate. After 24 hrs, gently remove themedia (containing planktonic cells) and gently wash each well 1-2 timeswith 2.0 ml of sterile PBS. Remove the wash. Gently deposit 1.0 ml offresh liquid growth media to each well. Gently deposit 1.0 ml water(control) or testing agent to each well. Incubate plate at 32° C.,rocking at 15 rpm for another 24 hours. Gently remove themedia/planktonic cells and gently wash each well twice with 2:0 mlsterile PBS. Remove the wash. Add 2.0 ml of fresh sterile PBS to eachwell. Sonicate the 12 well plate for 15 seconds. Remove the biofilmcontaining PBS solution, scrapping the bottom of the plate with thepipette tip and place in a sterile 15 ml tube. Vortex tubes for 30seconds. 200 l of solution was transferred from the tubes to a 96 wellplate where a serial dilution was done. The dilution series was thenplated in duplicate onto mDixon plates. Biofilm-embedded viable cellswere quantified by colony forming unit (CFU) counts after 4 days ofincubation at 32° C. The experiment was repeated twice and the resultsare combined. The combination of NaEDTA (0.5 mg/ml) and S. cit (3.2mg/ml) performed significantly better than NaEDTA at 0.5 mg/ml(p=0.0129) or sodium citrate alone (p=0.00007), as shown in FIG. 25.FIG. 25 also shows that the combination of NaEDTA (0.5 mg/ml) and S. cit(6.4 mg/ml) performed significantly better than NaEDTA at 0.5 mg/ml(p=0.0054) or sodium citrate alone (p=0.00002).

We claim:
 1. A composition for inhibiting biofilm formation caused byMalassezia pachydermatis, Staphylococcus aureus, or Malassezia furfur,the composition comprising: (a) EDTA salt at a concentration of about0.5 mg/ml of the composition; and (b) sodium citrate at a concentrationof between about 3.2 mg/ml to about 6.4 mg/ml of the composition.
 2. Thecomposition of claim 1, wherein the EDTA comprises disodium ortetrasodium EDTA.
 3. The composition of claim 1, comprising: about 0.5mg/ml EDTA salt and about 3.2 mg/ml sodium citrate.
 4. The compositionof claim 1, comprising: about 0.5 mg/ml EDTA salt and about 6.4 mg/mlsodium citrate.
 5. The composition of claim 1, further comprising one ormore ingredients selected from the group consisting of: water, a buffer,a stabilizing agent, a gelling agent, a surfactant, a herbal, a vitamin,a mineral, an extra cellular matrix, an antimicrobial, an antibiotic,and a pH adjuster.
 6. The composition of claim 1 prepared as one or moreof a disinfecting solution, a dip solution, a lotion, a cream, anointment, a gel, a spray, a dressing, a gauze, a bandage, athermoreversible gel spray, a wrap, an adhesive, a tape, a soak, ashampoo, and a balm.
 7. The composition of claim 1, wherein thecomposition is delivered using a liposome or nanoparticle.
 8. Thecomposition as claimed in claim 1, further comprising an anti-infectivecompound selected from the group consisting of alginate lyase, nisin,lactoferricin, serotransferrin, ovotransferrin, ovalbumin, ovomucoid,protamine sulfate, chlorhexidine, cetylpyridinium chloride, triclosan,silver sulfadiazine, benzalkonium chloride, hydrogen peroxide, citricacid, potassium citrate, 5-fuorouracil, cis-2-decenoic acid, DNase I,proteinase K, silver, gallium, bacteriocins, antimicrobial peptides andan enzyme that cleaves poly-B-1,6-N-acetylglucosamine.
 9. A method ofpreventing or treating wound infection, comprising topical ornon-topical application of the composition of claim 1, wherein the woundinfection is selected from one or more of infections of cuts, bruises,surgical sites, lacerations, abrasions, punctures, incisions, gunshots,burns, pyoderma, otitis media, otitis externa, otitis interna, cow uddermastitis, atopic dermatitis, eczema, pressure ulcers, venous and arteryleg ulcers, and diabetic foot ulcers.
 10. The method as set forth inclaim 9, further comprising multiple applications of the composition.11. The method as claimed in claim 9, wherein the method is used totreat one or more of humans, domestic animals, farm animals, zooanimals, pet animals, dogs, horses, cats, cattle, pigs, goats and sheep.12. A method of preventing or treating meat spoilage comprising topicalor non-topical use of the composition of claim 1 on meat or meatproducts.
 13. A method of disinfecting meat comprising topical use ofthe composition of claim 1 on meat or meat products.
 14. The method ofclaim 12, wherein the meat or meat products comprises intestine orintestinal parts of pigs, cattle, sheep, goats or horses used for makingsausage casings.
 15. A method of preventing or treating meat spoilagecomprising topical or non-topical use of the composition of claim 1 onor impregnated into collagen or cellulose for making artificial sausagecasings.
 16. The method of claim 12, wherein the topical or non-topicaluse comprises one or more of coating, spraying, misting, injecting,soaking, flushing, dipping and rinsing.
 17. A composition for inhibitingbiofilm formation caused by Malassezia pachydermatis, Staphylococcusaureus,or Malassezia furfur, the composition comprising an antimicrobialagent consisting of: (a) EDTA salt at a concentration of about 0.5 mg/mlof the composition; and (b) citrate salt at a concentration of betweenmore than about 3.2 mg/ml to about 6.4 mg/ml of the composition.